Crystallization

Facilities and equipment

Temperature controlled laboratory (22 oC) with a cold room dedicated for protein crystallization is located at the Faculty of Biochemistry and Molecular Medicine at the Kontinkangas campus of University of Oulu. The protein crystallization laboratory is equipped with TTP Lab Tech Mosquito LCP nanodispenser for setting-up crystallization experiments, TECAN pipetting robot for liquid handling, two Formulatrix Rock Imagers for imaging of the crystallization experiments and several microscopes for inspection of crystallization drops. The Zeiss Discovery V8 microscope with a motorized stage and a CMOS camera connected to a PC is for documentation of the crystallization results. Dedicated areas, equipment and tools are available for crystal storage, handling, shipment to synchrotrons and in house data collection.

Figure. Zeiss stereomicroscope with a camera for documentation of protein crystallization results.

Sample requirements

Protein sample intended for crystallization has to be pure and preferably not in phosphate buffer. The buffer concentration should be around 25 mM without any additional salt. Protein concentration should be approximately 5 mg/ml. The minimum required quantity for one screen with 96 crystallization conditions with 50 nl drop volume is 8 μL (40 µg protein in total).

Screening initial crystallization conditions

As a standard procedure, the initial crystallization conditions are screened with a sitting-drop vapor-diffusion method in a 96-well plate using crystallization screens made in-house or using commercial screens. The protein solution is mixed in different ratios with the crystallization solutions in a 100-200 nl -drop volume using a TTP Lab Tech Mosquito LCP nanodispenser available at the protein crystallization laboratory (F028). Hanging-drop vapor-diffusion as well as microbatch under oil method in 96-well format can also be used for initial screening. It is recommended to setup the crystallization experiments at room temperature as well as at 4 oC. Currently, we have the Factorial screen 1&2 (Oulu), PEG Smear (Oulu), Crystal Screen I&II (Hampton), ProPlex (Molecular Dimensions), JCSG-plus (Molecular dimensions), PACT premier (Molecular Dimensions) and Helsinki Cryo for initial screening. These screens, resulting in more than 1000 starting conditions, are documented on Wiki of Protein Crystallography accessible to local users.

Optimization

If there are crystals or “promising” precipitate the crystallization conditions are optimized varying the pH and the precipitant concentration around the condition of the hit. The optimization screen is prepared with the CrysScreen software and pipetted with the TECAN Freedom EVO liquid handling robot. Stock solutions are available for the in-house screens. If a suitable crystallization condition has been found, it is also worthwhile to optimize the condition using the hanging-drop (or sitting-drop) vapor diffusion method in 24-well plates and bigger drops (1 μL + 1 μL) from which the crystal mounting is easier and the crystals tend to grow to a larger size. Macro and micro seeding methods can also be used.

Crystallization plates

Crystallization experiments are set-up into a 96-well or a 24-well crystallization plates depending on the experiment (screening, optimization, soaking, hanging drop, sitting drop). For the sitting-drop vapor diffusion set-ups we are using the TTP Lab Tech Triple Sitting Drop plate, Corning 3556 plate and the Greiner 609101 CrystalQuick Crystallization plate. The microbatch under oil screen, where a small volume of protein solution and the crystallization solution are pipetted underneath an oil layer, are either carried out using the Douglas microbatch plate using the Tecan robot (0.5-1 µl) or Swissci 3 well plate (Hampton Research) using Mosquito pipetting robot (50-1200 nl).

The 7 screens available are (In Wiki, scroll down the page to Crystallization screens):

There is more information on our crystallization setup on our local Wiki.

Imaging

The crystallization experiments on the 96-well plates are imaged with Formulatrix Rock Imagers, RI54 and RI27. RI54 is located at 22 oC and RI27 is in a cold room at + 4 oC. RI54 is equipped with a UV imaging option. Images of the crystallization experiments can be viewed via a web browser using the xtalPiMS software which is being developed in collaboration with Diamond Light Source (Oxford, UK) and Instruct for the viewing and documentation of the crystallization experiments and for data tracking of protein crystal handling.